Neural tube defects (NTDs) are among the most common of congenital defects, with prevalence rates ranging from 1 per 1000 to 8 per 1000 births depending on ethnic group. The differential prevalence among ethnic groups is consistent with other date suggesting that there are genetic factors associated with NTDs. Such an association is confirmed by animal studies showing that susceptibility to developmental toxicant-induced NTDs varies among different inbred strains of mice. Both human and animal studies suggest that the genetic component of NTDs is complex and likely to involve multiple loci. In addition, a variety of drugs, chemicals and physical agents are known to increase the incidence of NTDs in animals and in some cases humans. Despite the prevalence of NTDs and their associated toll on infants with NTDs and their families, the etiology of NTDs remains poorly understood despite concerted effort over the past 20 years. The lack of understanding is due, in part, to the complexity of NTDs and to the lack, until recently, of experimental approaches that allow a global analysis of patterns of gene and protein expression during complex disease processes like NTDs. Within currently funded grants, we are using DNA microarrays to assess global patterns of gene expression in embryos exposed to hyperthermia. By using embryos from strains of mice that are either sensitive or resistant to hyperthermia-induced exencephaly, we hope to identify specific genes associated with hyperthermia-induced NTDs. In this grant, we hypothesize that recent developments in proteomics, specifically quantitative analysis of complex protein mixtures using isotope-coded affinity tags (ICATS analysis) and the analysis of protein phosphorylation, will provide significant and exciting new methods to study the role of protein in the etiology of hyperthermia-induced exencephaly. Using these new proteomics technologies, we propose to use mouse strains differentially sensitive to hyperthermia-induced exencephaly to identify proteins and their associated pathways that are modified during hyperthermia teratogenesis. To accomplish this within the context of a Developmental Toxicology Exploratory (R21) Research Grant, we set forth the following specific aims: Specific Aim 1: compare protein expression profiles in mouse embryos of the SWV (sensitive) and C57BL/6 (resistant) strains of mice after exposure to hyperthermia (43 degrees C) on day 8.5. This comparison will be accomplished by undertaking a quantitative analysis of complex embryo protein mixtures using isotope-coded affinity tags (ICAT). Specific Aim 2: Compare phosphoprotein expression profiles in mouse embryos of the SWV (sensitive) and C57BL/6 (resistant) strains of mice after exposure to hyperthermia (43 degrees C) on day 8.5. This comparison will be accomplished using a newly-developed systematic approach to the analysis of protein phosphorylation in complex protein mixtures.